Long-lasting precast electrophoresis gel

Abstract

Disclosed is a precast polyacrylamide gel for use in gel electrophoresis, comprising polyacrylamide and an aqueous Tris 0.04 M to 0.15 M solution, at least one first ampholyte exhibiting an isoelectric point (pI) of from 5.4 to 6.4 at a total concentration of from 0.01 M to 0.4 M; and at least one second ampholyte exhibiting an isoelectric point (pI) of from 2.5 to 3.5 to adjust the pH to between 5.5 and 7.5 at the temperature of 25° C. The precast gels can be used for electrophoresis of oligopeptides, polypeptides, oligonucleotides, and polynucleotides under denaturating or nondenaturing conditions, and exhibit long shelf-life.

1 . A precast polyacrylamide gel for use in gel electrophoresis, comprising polyacrylamide and:
 i) an aqueous solution of tris(hydroxymethyl)aminomethane (Tris) at a concentration of from 0.04 M to 0.15 M;   ii) at least one first ampholyte exhibiting an isoelectric point (pI) of from 5.4 to 6.4 at a total concentration of from 0.01 M to 0.4 M; and   iii) at least one second ampholyte exhibiting an isoelectric point (pI) of from 2.5 to 3.5 to adjust the pH to between 5.5 and 7.5 at the temperature of 25° C.   
     
     
         2 . A precast gel according to  claim 1 , comprising:
 i) an aqueous solution of Tris at a concentration of from 0.4 M to 0.15 M;   ii) at least one first ampholyte exhibiting an isoelectric point (pI) of from 5.4 to 6.4 at a total concentration of from 0.01 M to 0.4 M; and   iii) at least one second ampholyte exhibiting an isoelectric point (pI) of from 2.5 to 3.5 to adjust the pH to between 6.0 and 7.0 at the temperature of 25° C.   
     
     
         3 . A precast gel according to  claim 1 , comprising:
 i) Tris at a concentration of 0.06 M to 0.10 M;   ii) at least one first ampholyte selected from the group consisting of glycine, serine, asparagine, tryptophane, methionine, and phenylalanine at a total concentration of from 0.1 M to 0.35 M;   iii) at least one second ampholyte selected from the group consisting of aspartic acid and glutamic acid to adjust the pH to between 6.2 and 6.8 at the temperature of 25° C.; and   iv) polyacrylamide at a concentration of from about 4 w/v % to about 20 w/v %.   
     
     
         4 . A gel according to  claim 1 , wherein said first ampholyte comprises asparagine and glycine at a total concentration of from 0.2 M to 0.35 M. 
     
     
         5 . A gel according to  claim 1 , wherein said first ampholyte comprises asparagine and serine at a total concentration of from 0.2 M to 0.35 M. 
     
     
         6 . A gel according to  claim 1 , comprising polyacrylamide at a concentration of from about 4 w/v % to about 20 w/v %. 
     
     
         7 . A gel according to  claim 1 , comprising Tris at a concentration of 0.06 to 0.1 M, at least one first ampholyte selected from asparagine, serine, glycine, tryptophane, methionine, and phenylalanine at a total concentration of from 0.15 to 0.35 M, and at least one second ampholyte selected from aspartic acid and glutamic acid to adjust the pH of from 6.2 to 6.8. 
     
     
         8 . A precast gel according to  claim 1 , comprising
 i) polyacrylamide at a concentration of from about 4 w/v % to about 20 w/v %;   ii) an aqueous solution of tris(hydroxymethyl)aminomethane (Tris) at a concentration of 0.06 to 0.10 M;   iii) at least one first ampholyte exhibiting an isoelectric point (pI) of from 5.4 to 6.4 at a total concentration of from 0.15 to 0.35 M; and   iv) at least one second ampholyte exhibiting an isoelectric point (pI) of from 2.5 to 3.5 to adjust the pH to between 6.2 and 6.8 at the temperature of 25° C.   
     
     
         9 . A gel according to  claim 1  for use in gel electrophoresis under denaturation conditions. 
     
     
         10 . A gel according to  claim 1 , for use in SDS-PAGE. 
     
     
         11 . A gel according to  claim 1  for use in gel electrophoresis under nondenaturation conditions. 
     
     
         12 . A gel according to  claim 1 , further optionally comprising at least one component selected from surfactants, solvents, electrolytes, denaturation agents, and dyes. 
     
     
         13 . A precast gel according to  claim 1 , comprising
 i) polyacrylamide at a concentration of from about 4 w/v % to about 20 w/v %;   ii) an aqueous solution of tris(hydroxymethyl)aminomethane (Tris) at a concentration of 0.06 to 0.10 M;   iii) at least one first ampholyte exhibiting an isoelectric point (pI) of from 5.4 to 6.4 at a total concentration of from 0.15 to 0.35 M;   iv) at least one second ampholyte exhibiting an isoelectric point (pI) of from 2.5 to 3.5 to adjust the pH to between 6.2 and 6.8 at the temperature of 25° C.; and   v) one or more components selected from surfactants, solvents, electrolytes, denaturation agents, and dyes.   
     
     
         14 . A precast gel according to  claim 1 , having a shelf life of at least 12 months. 
     
     
         15 . A method of preparing a stable, high performance gel for electrophoresis of biomolecules, comprising
 i) providing an aqueous solution comprising from acrylamide (AA) and bis-acrylamide; from 0.05 to 0.15 M Tris; at least one ampholyte exhibiting an isoelectric point (pI) of from 5.4 to 6.4 at a total concentration of from 0.01 to 0.4 M; and at least one ampholyte exhibiting an isoelectric point (pI) of from 2.5 to 3.5 to adjust the pH between 5.5 and 7.5 at the temperature of 25° C.;   ii) adding into the mixture obtained in step i) an aqueous solution of ammonium persulfate while mixing, and adding tetramethylethylenediamine (TEMED) while mixing;   iii) carefully homogenizing the mixture obtained in step ii) without trapping air bubbles;   iv) casting the mixture obtained in step iii) into a slab-shape of desired dimensions; and   v) sealing the slab-shaped gel in a polymeric wrap.   
     
     
         16 . A method according to  claim 15 , wherein the concentration of the acrylamide (AA) and bis-acrylamide is from 4 to 20 w/v %. 
     
     
         17 . A method according to  claim 15 , comprising i) providing aqueous solution comprising AA:Bis, Tris at a concentration of 0.05 to 0.15 M, at least one first ampholyte selected from glycine, serine, asparagine, tryptophane, methionine, and phenylalanine at a concentration of from 0.15 to 0.35 M, and at least one second ampholyte selected from aspartic acid and glutamic acid to a pH between 6.0 and 7.0; and ii) polymerizing said PAA, thereby obtaining a slab gel. 
     
     
         18 . A method according to  claim 15 , wherein said biomolecule is selected from oligopeptides, polypeptides, oligonucleotides, and polynucleotides. 
     
     
         19 . A method according to  claim 15 , wherein said biomolecule is selected from proteins and nucleic acids.